Jurado-Cobena Eduardo, Ikegami Tetsuro
Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, Galveston, TX, USA.
Department of Pathology, The Sealy Institute for Vaccine Sciences, The Center for Biodefense and Emerging Infectious Diseases, The University of Texas Medical Branch at Galveston, Galveston, TX, USA.
Methods Mol Biol. 2025;2893:273-283. doi: 10.1007/978-1-0716-4338-9_20.
Oropouche fever, a mosquito- or midge-borne emerging zoonotic disease endemic to South and Central America, manifests as a dengue-like acute febrile illness with occasional occurrences of meningitis or meningoencephalitis. The causative agent, Oropouche virus (OROV), belongs to the genus Orthobunyavirus within the family Peribunyaviridae. Its tripartite negative-sense RNA genome comprises small (S), medium (M), and large (L) segments, encoding structural N, Gn/Gc, and L proteins, respectively. Additionally, the S- and M-segments encode nonstructural proteins: NSs and NSm, which may act as virulence factors. OROV NSs functions as an interferon antagonist with an unknown mechanism, while the roles of OROV NSm remain elusive. This chapter introduces efficient expression systems for OROV NSm and NSs proteins. Validating the presence of a signal peptide at the N-terminus of NSm protein is essential for its expression. Furthermore, expressing OROV NSs protein independently of an RNA polymerase II promoter is crucial to prevent restricted gene expression, potentially caused by NSs inhibiting cellular RNA polymerase II, as observed in closely related bunyavirus NSs proteins. These protein expression strategies offer insights into the molecular characterization of OROV NSm and NSs proteins, facilitating a deeper understanding of their virulence mechanisms.
奥罗普切热是一种由蚊子或蠓传播的新发人畜共患病，在南美洲和中美洲流行，表现为类似登革热的急性发热性疾病，偶尔会出现脑膜炎或脑膜脑炎。病原体奥罗普切病毒（OROV）属于白蛉病毒科正布尼亚病毒属。其三部分负链RNA基因组由小（S）、中（M）和大（L）片段组成，分别编码结构蛋白N、Gn/Gc和L。此外，S和M片段编码非结构蛋白：NSs和NSm，它们可能作为毒力因子。OROV NSs作为一种干扰素拮抗剂，作用机制尚不清楚，而OROV NSm的作用仍不明确。本章介绍了OROV NSm和NSs蛋白的高效表达系统。验证NSm蛋白N端信号肽的存在对其表达至关重要。此外，独立于RNA聚合酶II启动子表达OROV NSs蛋白对于防止可能由NSs抑制细胞RNA聚合酶II引起的基因表达受限至关重要，这在密切相关的布尼亚病毒NSs蛋白中已有观察。这些蛋白质表达策略为OROV NSm和NSs蛋白的分子特征提供了见解，有助于更深入地了解它们的毒力机制。
